The long term objective of our research is the elucidation of the molecular mechanisms of the acute and chronic effects of opiate narcotic analgesics. We are particularly interested in the role played in opiate actions by the endogenous opioid system. Knowledge of the functioning of this neuropeptide system should ultimately have wide-ranging implications in the treatment of drug abuse and chronic pain. The focus of our research is on opioid receptor structure and function, with emphasis on receptor regulation at the gene, posttranslational, second messenger and physiological levels. The mu-opioid binding protein, previously purified to homogeneity in our laboratory, has now been reconstituted to yield stereospecific, selective, high affinity mu-agonist binding. Studies on the second messenger systems coupled to the mu receptor and on receptor regulation by phosphorylation in whole cells and in the reconstituted system are proposed. These studies in cells and tissues will be extended to other receptor types and subtypes, as will the studies on reconstituted systems, when purified or recombinant receptors become available. Structural studies will be done on the nature of the carbohydrate portion of the receptor glycoproteins, the lipids critical for function and the functional domains (ligand binding, G-protein coupling, etc.) of the receptor protein. Biochemical methods will be complemented by molecular biological techniques, such as substitution and deletion analysis and site-directed mutagenesis. The effects of physiological and pharmacological parameters, such as aging, chronic antidepressants and food deprivation will be studied with respect to changes in properties and number of the different opioid receptor types and, where appropriate, with respect to receptor gene regulation. The latter will be done using the techniques of Northern blot analysis and solution hybridization. When changes are observed, they will be mapped to specific brain regions by in situ hybridization. Efforts to clone the mu receptor and other opioid receptor types and subtypes will continue.